This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/FI98/00445 which has an International filing date of May 28, 1998, which designated the United States of America.
This invention describes a DNA construct based on viral sequences which are capable of activating or increasing the expression of a gene in recombinant DNA-containing tissue. The invention is useful for increasing the expression of a gene, derived from heterologous plant species, or has non-plant origin. The invention will facilitate in the genetic engineering to super-express the proteins of interest or express novel plant phenotypes of economic or investigative value.
This invention is related to molecular biology and biotechnology exploiting plant genetic engineering by recombinant DNA technology.
Structurally polycistronic RNAs of many plant and animal viruses belonging to so-called Sindbis-like supergroup are functionally monocistronic: only the 5xe2x80x2-proximal gene can be translated by eukaryotic ribosomes. All the internal translationally silent genes are expressed from subgenomic RNAs (sgRNA) produced by transcription of the minus-copy of the full-length genomic RNA from internal sitesxe2x80x94subgenomic promoters (sgPr). All RNA viruses produce during their replicative cycle the virus-specific RNA-dependent RNA polymerase (replicase) which is essential for the synthesis of various species of viral RNA. The replicase gene is localized 5xe2x80x2-proximally within the monopantite genomes of the members of Sindbis-like viruses (e.g. tobacco mosaic virus, TMV, potato virus X, PVX, brome mosaic virus, BMV), i.e. represent the onlv translatable gene of the polycistronic genome (for a review, see Bruening et al. (1979) In xe2x80x9cMolecular Biology of Plantsxe2x80x9d, Academic Press, New York-London. pp.241-272). Specific cis-acting sequences within the minus-copy of the genomic RNA are required for synthesis of sgRNAs. The multifunctional nature of replicase allows it to recognize these internal sequences (sgPrs) to synthesize sgRNAs by partial transcription of the negative-scrand RNA. This mechanism has been clearly established for some plant viruses in vitro (Miller et al. (1985) Nature 313, 68-70) and in vivo (Gargouri et al. (1989) Virology 171, 386-393).
It has been shown that the chimeric TMV (Donson et al. (1991) Proc.Natl.Acad.Sci.USA 88, 7204-7208) and PVX (Chapman et al. 1992) Plant J. 2, 549-557; Hammond-Kosack et. al. (1995) Mol.Plant-Microbe Int. 8, 181-185) vectors could be constructed by insertion of the foreign genes downstream of a sgPr that permits the expression of introduced genes from appropriate sgRNA. This means that viral replicase can recognize the sgPr at different positions within recombinant minus-strand. Moreover, viral replicase expressed from the integrated cDNAs in transgenic plants can replicate viral RNAs and produce subgenomic RNAs (Leiser et al. (1992) Proc.Natl.Acad.Sci.USA 89, 9136-9140). Thus, it could be presumed that the replicase will be able to act in trans to recognize in vivo the specific sgPr not only in full-length viral genome but also in the short chimeric minus-sense RNA transcripts carrying a foreign gene (in antisense orientation). This could result in producing the respective sgRNA by the mechanism adapted for viral sgRNA synthesis.
Contrary to the majority of eukaryonic mRNAs several viral and cellular mRNAs are translated by alternative-internal ribosome entry mechanism that bypasses the normal cap recognition step and 5xe2x80x2-nontranslated sequence scanning. In particular, the genome of crucifer tobarnoviruses (crTMV) (Dorokhov et al. (1994) FEBS Letters 350, 5-8) contains two cis-acting sequences mediating internal ribosome entry and translation of the 3xe2x80x2-proximal genes of crTMV RNA. These elements can be used in constructing functionally dicistronic or polycistronic eukaryotic mRNAs. In this invention we found that these elements can be expoited in certain conditions for the expression at translational level of more than one reporter gene within a polycistronic mRNA in eukaryotic cells.
The complete nucleotide sequence of the PVX genome has been reported for Russian (Skryabin et al. (1988a) Nucleic Acid Res. 16, 10929-10930) and several other strains (for example, see Querci et al. (1993) J.Gen.Virol. 74, 2231-2255.). The PVX genome contains five ORFs coding for the 165 kDa replicase, three movement proteins (MPs) (25 kDa, 12 kDa and 8 kDa) and coat protein (CP) (FIG. 1). The replicase protein is translated directly from the genomic RNA, and its expression is controlled by the 5xe2x80x2-untranslated genomic leader sequence (xcex1xcex2-sequence). The xcex1xcex2-leader has been shown to enhance strongly the translation of foreign genes both in vitro (Smirnyagina et al. (1991) Biochimie 73, 587-598) and in vivo Tomashevskaya et al. (1993) J.Gen.Virol. 74, 2717-2724). The separate sgRNAs are produced in PVX infection for the MPs and CP expression which are 3xe2x80x2-coterminal with the genomic RNA (FIG. 1) (Morozov et al. (1991) J.Gen. Virol. 72, 2039-2043). The precise borders of the PVX sgPrs are unknown, however, it has been experimentally shown that the 81 nt sequence including 15 5xe2x80x2-terminal nucleotides of the PVX CP gene and 66 nt upstream sequence is active in vivo as sgPr (Chapman et al. (1992) Plant J. 2, 549-557). Recently the PVX-based vectors with this engineered 81-nt-long sgPr was used for the transient expression of the pathogene elecitor gene and plant defence genes (Rommens et al. (1995) Plant Cell 7, 249-257). Using of plant RNA viruses for the introduction and expression of non-viral foreign genes in plants has been demonstrated by the cited references above as well as by French et al. (1986) (Science 231, 1294-1297). However, all these viral vectors have been capable of autonomous replication in plant cells, thus, providing a risk for cell pathogenesis in a manner typical for wild type virus. Another disadvantage of self-replicating RNA vectors is that they are not stable for the maintenance of non-viral sequences (Donson et al. (1991) Proc.Natl.Acad.Sci.USA 88, 7204-7208).
While there are distinct needs for improving expression of foreign proteins, such as industrial enzymes, medical drugs and so on in plants, there exists several technical problems. First of all, the expression levels of protein in plants are not adequate. The present invention was aimed to overcome these drawbacks.
The present invention focuses on the super-expression of foreign genes in transgenic cells by to combining within a single cDNA construct and respective RNA transcript, several trans- and cis-acting genetic elements of viral origin which will act in concert to trigger the following functional events: a) the primary chimeric continuous RNA transcript is produced by the transformed cells from plant-expressible promoter (35S promoter) (FIG. 2); b) RNA replicase produced by direct translation of the 5xe2x80x2-proximal gene of a single continuous primary transcript will synthesize secondary monocistronic (FIG. 3A) (or dicistronic (FIG. 3B)) mRNA as a result of the transcription from sgPr sequence. Expression of the 5xe2x80x2-proximal gene of these mRNAs will be enhanced by the xcex1xcex2-leader. Translation of the 5xe2x80x2-distal gene of dicistronic mRNA (FIG. 3B) will be promoted by internal ribosome entry site (IRES) sequence derived from crTMV tobamovirus mentioned above; c) it is probable that at least part of RNA transcripts originated from sgPr will include at their 3xe2x80x2-end the minus copy of RNA replicase gene and genomic promoter for plus-RNA synthesis (FIG. 3A and B). It can be expected that RNA replicase produced in transgenic cell will bind with the 3xe2x80x2-terminal sequence of this RNA (genomic promoter) producing upon transcription the RNA molecules carrying the plus-polarity replicase gene at the 5xe2x80x2-end. Translation of these mRNAs will result in production of additional replicase in transgenic plant (FIG. 4).